Functional features of the collectins and ficolins: Activation of the complement

Aiming the elimination of microorganisms, the collectins cooperate with phagocytes and humoral factors, including the complement.

The complement system is a defence mechanism activated by innate and acquired immune mechanisms. It consists of serum proteins together with an associated group of cell membrane proteins. When complement proteins bind to the surface of microorganisms, enzymatic pathways are activated, and in the next step microorganisms are destroyed. In healthy animals, these pathways are inactive, but they can be evoked either by the presence of antibodies on the surface of organisms or by recognition of the complex carbohydrate moieties on the surface of infectious agents (TIZARD 2008). The complement system consists of proteins, which are either labelled numerically with the prefix C (for example C1 or C2) or designated by letters of the alphabet. The complement proteins are mainly synthesised in the liver (C3, C6; C8 and B), but some are also made by macrophages (C2, C3, C4, C5, B, D, and P). A number of the proteins can be found free in the serum, whereas others are cell-bound.

The complement cascade can be activated through three distinct pathways, which are named the classical, alternative and lectin pathways.

The activation of the classical pathway requires pre-sensitisation of microorganisms with antibodies, and is thus part of the acquired immune system. The first component of this pathway is a multimolecular protein complex called C1, which consists of three

proteins: C1q, C1r and C1s. C1 can be described as a complex of C1q, which is associated with C1s and C1r. C1q is able to recognize the Fc region of IgG and IgM immunoglobulins bound to an antigen. Subsequently recognition leads to the formation of a membrane attack complex (MAC) and microbial lysis. This occurs via the activation of several complement components and the initiation of a potent protease (C3 convertase).

In contrast to the classical pathway, both, the alternative and the lectin pathway are independent of clonal immunity and for that reason parts of innate immunity.

The alternative pathway is triggered when microbial cell walls come into contact with complement components in the bloodstream. The complement protein C3 plays a key role within this pathway, as it binds to acceptor groups on many pathogens and marks them for destruction by immune cells.

C3 spontaneously breaks down into two fragments called C3a and C3b, even under normal conditions in healthy animals. This process is called tick-over. C3b is constructed of a highly reactive carbonyl group, which irreversibly binds to the target cell surface. The target cell is not necessarily a pathogen, such as a bacterium, but can also be a normal cell of the body. To avoid activation of the alternative pathway by normal cells, the further activity of C3b is controlled by two factors, named factor H and I. Factor H interacts with normal cell surfaces and glycoproteins rich in sialic acid and other neutral or anionic polysaccharides, resulting in the destruction of C3b by the activation of factor I. Consequently in a healthy individual, factors H and I destroy C3b as fast as it is generated. Due to the fact that the components of bacterial cell walls, such as lipopolysaccharides (LPS), lack sialic acid, factor H cannot bind to C3b. Factor I remains inactive, and the C3b persists. Subsequently the pathogen is marked by C3b and accessible for recognition by phagocytes, which then destroy the cell. The process of marking a pathogen, in this case with a complement factor, is called opsonisation. Furthermore, also the formation of a membrane attack complex (MAC), which inserts itself into a microbial cell membrane resulting in osmotic lysis of the pathogen, can be triggered by bound C3b. Binding of C3b leads to activation and cleaving of several other complement factors, in which factor C5 plays an important role (JANEWAY et al. 2005; TIZARD 2008).

In our study the lectin pathway is of major interest, as it is activated by MBL and also by ficolins, which bind to the surface sugar residues on microorganisms (IKEDA et al. 1987; HOLMSKOV et al. 1994; EPSTEIN et al. 1996; LU 1997; MATSUSHITA and FUJITA 2001; VAN DE WETERING et al. 2004; PHANEUF et al. 2007).

The discovery that MBL activates complement independently of antibodies was made in 1987 by IKEDA et al. (1987). This finding was noticed to be a major breakthrough for the understanding of its role in innate immunity (HOLMSKOV et al. 2003).

The ligands for the collectins are bacterial cell wall components like lipoteichoic acid of Gram-positive and LPS of Gram-negative bacteria (POLOTSKY et al. 1996;

HOLMSKOV et al. 2003; GUPTA and SUROLIA 2007).

As collectins coat microorganisms, they act as opsonins. Subsequently specific interaction between the collectins and receptors on phagocytic cells may result in increased association, uptake and killing of the pathogens (MCNEELY and COONROD 1994; PIKAAR et al. 1995; OFEK et al. 2001). In case of MBL it either opsonises microorganisms directly or it can lead to opsonisation through complement activation and deposit of complement factor C3 (PETERSEN et al. 2001;

HOLMSKOV et al. 2003).

MBL circulates in complex with serine proteases, known as mannan-binding lectin associated serine proteases (MASPs). Three types of MASPs have been indentified until now: MASP-1 (MATSUSHITA and FUJITA 1992), MASP-2 (THIEL et al. 1997) and MASP-3 (DAHL et al. 2001). If MBL binds to the surface of microorganisms, MASPs are activated to recruit and cleave complement factors. MASP-2 has been shown to cleave C4 and C2 (THIEL et al. 1997). By the splitting of C4 and C2 a cell-bound complex called C4bC2b is generated, which is able to activate the C3 convertase. As a result, great amounts of C3b and C3a are created. C3a acts as a peptide mediator for inflammatory cells, whereas C3b binds the surface of microorganisms and functions as an opsonin, resulting in cell marking for destruction through phagocytes. Furthermore, C3b attaches to C5 and the C5 convertase is generated. It subsequently cleaves C5 into C5a and C5b. C5a also, like C3a, is an important mediator for inflammatory cells. C5b functions as an activator for the synthesis of the MAC (JANEWAY et al. 2005; TIZARD 2008). The MASP-1 is responsible for the direct cleavage of C3 (MATSUSHITA and FUJITA 1995). The function of MASP-3 still remains unclear, and no relevant substrate for this serine protease has been identified by now (GÁL et al. 2007).

Also ficolins activate the lectin complement pathway, by association of the molecules with MASPs (MATSUSHITA et al. 2000; RUNZA et al. 2008).

It needs to be emphasised that the lectin pathway and the classical pathway are not completely isolated from each other; neither in structure of the activating molecules, nor in function (FUJITA et al. 2004).

The lectin-MASP complex is structurally and functional equivalent to C1, which is composed of the serine proteases C1r and C1s and the recognition subcomponent C1q. This complex represents the first component of the classical complement pathway.

In the MBL pathway, MASP-2 is the enzyme part, like C1s in the classical pathway, which cleaves the complement components C4 and C2 to form the C3 convertase C4bC2b (THIEL 1992; THIEL et al. 1997; FUJITA et al. 2004). Besides, MASP-1 is capable of cleaving C3 directly (DAHL et al. 2001; ROSSI et al. 2001), with a resulting activation of the alternative pathway (MATSUSHITA and FUJITA 1995).

By means of structure, C1q and MBL also show great similarity. As already described previously, MBL is organised in form of a flower bouquet, consisting if six trimeric subunits. The structure of C1q consists of six globular heads, each connected by a strand to a central fibril-like region, composed of collagen-like triple-helical structure (KISHORE and REID 2000).

One difference between MBL and C1q structure results from the fact that MBL is composed of three identical polypeptide chains, respectively, whereas C1q is composed of three different chains (FUJITA et al. 2004)

However, it still can be resumed that the overall structure of C1q is similar to that of MBL and ficolins (HOLMSKOV et al. 2003; VAN DE WETERING et al. 2004).

Furthermore, the structure of MASP family members associated with MBL and ficolins resembles that of C1r and C1s, as they both consist of six domains, composed of proteins and proteases (FUJITA et al. 2004).